The papillomavirus origins of DNA replication and the E6/E7 promoters share overlapping sequences. In addition, transcriptional regulatory proteins have been shown to be required for replication and replication proteins modulate transcription. These regulatory regions have been analyzed in two viruses: human papillomavirus type 16 (HPV-16) and bovine papillomavirus (BPV-1). The HPV-16 long control region (LCR) contains three binding sites for the E2 transcriptional regulatory region. Mutational analysis of these sites demonstrated that there is a direct correlation between the number of E2 binding sites and the amount of DNA replication in transient assays in the SCC-9 squamous cell carcinoma cell line. In these assays the viral E1 and E2 proteins are expressed from separate expression vectors. Mutation of the P97 (E6/E7 promoter) TATA box had minimal effect on replication, indicating that transcription from this region is not required for replication. The corresponding region of BPV-1 contains several E2 binding sites as well as binding sites for cellular transcription factors including SP1, E2F, AP-1, YY-1, and NF-1. Mutations were made in these sites in the context of a P89 (E6/E7) promoter reporter plasmid. The effects of these mutations were assayed by transient transfection in bovine embryo fibroblasts (BEF) in the presence or absence of the E2 protein. We have previously shown that three SP1 sites are required for both basal and transactivated expression. Similar results were seen for the NF-1 site. The YY-1 and AP-1 sites also appear to be important for regulation of P89 transcription. However, we have been unable to demonstrate a role for the E2F site in our assays.